The ideal diagnostic and prognostic evaluation of premalignant and malignant lesions of the breast will require much more new information about the evolution of human breast cancer. Despite an enormous research effort, investigators have been largely unable to identify morphological, cellular, or molecular events which unequivocally lead to the development of this disease. Although several recently described events and factors appear to play roles in mammary neoplasia, they have with few exceptions been studied in the limited contexts of breast cancer cell lines, animal models of breast cancer, or small series of relatively advanced clinical lesions, making it difficult to generalize the significance of these findings to the development and progression of human disease. Our goal is to study the involvement of several known factors, and to identify new ones, in the novel context of a sequence of hyperplastic, dysplastic, and malignant clinical lesions carefully chosen to represent important stages in a hypothetical model of breast cancer evolution. Specifically, we propose: [1] to study by immunohistochemistry the expression of 10 separate markers (HER-2/neu, EGFR, TGF-alpha, RB, p53, PCNA/cyclin or topoisomerase II, cathepsin D, laminin receptor, urokinase- type plasminogen activator, and stromelysin-3) in each of 6 histologically distinct types of lesions hypothesized to represent sequential stages in the initiation/induction (benign typical and atypical proliferative disease), transformation (duct carcinoma in-situ), and progression ("early", "late", and metastatic invasive duct carcinoma) phases of breast cancer evolution; [2] to identify new markers of transformation and progression, using subtraction hybridization and differential screening of cDNA libraries prepared from samples of adjacent evolutionary stages, and to develop antibodies to these markers for use in histochemistry; and [3] to investigate the prognostic significance of selected markers during the progressive stages of "early", "late", and metastatic invasive duct carcinoma. The anticipated benefits of this research are that, by studying patterns of marker expression within our working model of breast cancer evolution, new insights will be gained regarding the development and progression of this disease and the biological significance of the markers being studied, and new information will emerge which will be useful in the diagnostic and prognostic evaluation of premalignant and malignant lesions of the breast. In addition, by using the powerful molecular techniques of subtraction hybridization and differential screening, new factors will be identified which are important in specific stages of breast cancer development.